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research-article |
Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli "Federico II," Complesso Universitario di Monte S Angelo, 80126 Napoli, Italy
ABSTRACT
Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.
bovine, female reproductive tract, gamete biology, oviduct, redox, sperm, spermatozoa, sperm motility and transport, sulfhydryls
In mammals, millions of spermatozoa are ejaculated into the female reproductive tract during coitus, yet only a small number reach the ampulla, the site of fertilization, with the competency to interact with female gametes. This reduction occurs at multiple, selective checkpoints in the female reproductive tract that only a small vanguard population of competent spermatozoa is able to overcome. Of spermatozoa that successfully enter the isthmus (lower oviductal tract), only a subpopulation is able to adhere to the epithelial cells lining its lumen [1]. Adhesion prolongs sperm life [1] by delaying capacitation until periovulatory signals (still poorly characterized) induce release of the spermatozoa [2]. Sperm-oviduct adhesion, in addition to providing this reservoir function [3], has been demonstrated in vitro to occur only in spermatozoa of preexisting high quality. As shown in several studies in different species, oviductal adhesion occurs only in spermatozoa characterized by intact acrosomes [4], uncapacitated status [2, 5–8], low internal free calcium content, and reduced membrane protein tyrosine phosphorylation [8–10], superior morphology [11], normal chromatin structure [12], and superior zona pellucida binding and fertilization competence [13, 14]. Although adhesion to the oviductal epithelium is a transient step, it is essential for successful fertilization; however, little is known about the nature of molecules involved in adhesion and release. The oviductal microenvironment is a dynamic and complex milieu; it is fine-tuned to cross talk with both gametes and embryos, and its composition in different regions changes during the phases of the estrous cycle, matching the metabolic requirements of spermatozoa, oocytes, and embryos. It is now well known that free radicals such as reactive oxygen species (ROS) influence oocytes, spermatozoa, and embryos and their environments. Protection from ROS is afforded by scavengers present in both male and female reproductive tract fluids, as well as in seminal plasma. Intra- and extracellular enzymatic and non-enzymatic antioxidants are well represented in the female tract in several species, and their ability to improve fertilization and embryonic development has been clearly demonstrated [15–17]. As a consequence, antioxidants have also been used successfully as supplements for embryo culture media to improve in vitro development [15, 18, 19]. Antioxidants may also modulate the functional activity of spermatozoa within the female reproductive tract. At reduced concentrations, ROS demonstrate physiological functions—triggering sperm protein tyrosine phosphorylation, capacitation, and the acrosome reaction—whereas at high concentrations ROS may cause a series of damages that include membrane lipid peroxidation, oxidation of protein sulphydryls, loss of motility and of fertilizing potential, and DNA fragmentation [20–23].
In cattle, the major antioxidants found in the oviductal fluid are catalase, superoxide dismutase, glutathione peroxidase, and reduced glutathione (GSH) [24–26]. Antioxidants such as catalase and GSH reach their maximal concentration in the bovine oviduct toward the end of the estrous cycle [26] (i.e., when spermatozoa selected and stored through the process of oviductal adhesion are released to migrate toward the fertilization site).
In the work described here, the effects of several antioxidants on the processes of in vitro sperm-oviduct adhesion, modulation of sperm surface sulfhydryls, and capacitation were examined in bovine species. Herein we report for the first time that thiol-reducing agents are involved in the release of spermatozoa adhering to the tubal epithelium in vitro. Overall, data support the hypothesis that thiol-reducing agents modulate the sulfhydryl-disulfide status of redox-sensitive sperm surface proteins.
BSA (fraction V), penicillamine, beta-mercaptoethanol, cysteine, dithiothreitol (DTT), reduced and oxidized glutathione (GSH and GSSG), dimetiltiourea, trolox, superoxide dismutase, catalase, lysophosphatidylcholine (LPC), 5-iodoacetamido-fluorescein (IAF), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC), and Hoechst 33342 were from Sigma Chemical Company (Milan, Italy); fetal calf serum (FCS), gentamycin, fungizone, HEPES, and sodium bicarbonate were from Gibco (Milan, Italy); and glutaraldheyde was from TAAB Laboratories (Rome, Italy). Reagents and water for preparation of saline and culture media were all cell-culture tested.
Oviducts were collected from a slaughterhouse (SO.ME.CA. S. Antonio, Abate, Italy) and transported to the laboratory in Dulbecco phosphate-buffered saline (PBS) supplemented with 50 µg/ml gentamycin at 4°C. Laminae of epithelial cells were squeezed from oviducts of single animals and were cultured in M199 supplemented with 50 µg/ml gentamycin, 1 µg/ml fungizone, and 10% FCS, as previously described [4]. Bovine oviductal epithelial cells (BOEC) were cultured in 10-cm petri dishes (Falcon; Becton Dickinson, Milan, Italy) for 24–48 h, and then were transferred into four-well, tissue culture dishes (Nunclon, Roskilde, Denmark) with round gelatin-coated, 12-mm, German glass cover slips on the well bottoms. Medium was exchanged with fresh medium every 48 h, and cell confluence was attained in 7–10 days. Monolayers were used within 24–48 h after attainment of cell confluence. Within each experiment, BOEC monolayers from a single individual were washed three times in Tyrode albumin lactate pyruvate medium (sperm-TALP) [27], modified as described in [28], and left in this medium until addition of sperm (1–3 h).
Frozen semen from three bulls (0.5-ml straws, approximately 40 x 106 spermatozoa/straw; Semen Italy, San Giuliano Saliceta, Modena, Italy) was used in all experiments. Straws were thawed in a water bath at 38°C for 30 sec; the semen was applied to a discontinuous (90/40) Percoll gradient with or without 10 µg/ml Hoechst 33342 and centrifuged for 30 min at 180 x g. The pellet was washed twice in 2 ml of BSA-free sperm-TALP by centrifugation at 180 x g for 5 min, and then was resuspended in 200 µl of the same medium. Aliquots of recovered sperm were assessed for concentration and percentage of motility with a hemocytometer on a microscope stage heated to 38.5°C.
Sperm suspension recovered after Percoll centrifugation was added to BOEC monolayers that had been cultured on round, gelatin-coated, 12-mm glass cover slips in NUNC four-well plates in 750 µl of sperm-TALP at a concentration of 2–3 x 106/well; the samples were incubated at 39°C, 5% CO2 in air, 95% humidity, for 60 min. At the end of co-incubation, all free swimming spermatozoa were removed by extensive washings with sperm-TALP or were collected for further analysis. The sperm fraction unable to adhere after 1 h of co-culture was referred to as "unbound sperm." After removal of unbound sperm, all wells with adhering spermatozoa were washed extensively with sperm-TALP. Experimental wells were treated with different concentrations of tested molecules for 30 min. At the end of treatment, sperm-oviductal co-cultures were washed to remove released spermatozoa, then were fixed and analyzed to quantify the number of adhering spermatozoa, as previously described [29]. Briefly, monolayers grown on gelatin-coated coverslips and inseminated with Hoechst-labeled spermatozoa were fixed in glutaraldehyde (2.5%) in PBS for 1 h at 20–25°C, extensively washed, and mounted with the same buffer on a glass slide with cells facing up. For each well, fields of 0.286 mm2 were acquired on a Zeiss Axioplan microscope equipped with phase-contrast, fluorescence, and Nomarsky optics, by means of an Optronix camera and KS 300 software (Zeiss, Milan, Italy). The number of adhering spermatozoa was determined by analyzing ten fields of 0.286 mm2 for each well.
Experiment 1. Experiment 1 (n = 5) was designed to study the effect of the thiol-reducing agents D- and DL-penicillamine, beta mercaptoethanol, and cysteine on release of spermatozoa adhering to oviductal monolayers. For this purpose, monolayers with adhering spermatozoa were incubated 30 min with sp-TALP alone (control for sperm release), or with thiol-reducing agents at 0.1 and 1 mmol/L in sp-TALP. At the end of treatment, wells were washed extensively with sp-TALP, then were fixed and analyzed as described below.
Experiment 2. Experiment 2 (n = 4) was performed to understand whether the sperm-releasing effect of penicillamine was exerted on spermatozoa or oviductal cells. To this end, spermatozoa recovered by Percoll centrifugation were divided into two aliquots that were preincubated for 30 min with 100 µM penicillamine or with sp-TALP alone, washed in sp-TALP by centrifugation at 200 x g for 10 min, and subsequently inseminated at 2 x106 motile spermatozoa/well in a final volume of 0.5 ml. Some monolayers were treated similarly and washed extensively to remove penicillamine completely. The final concentration of penicillamine in the wells inseminated with pretreated spermatozoa was 2 µmol/L. Parallel control wells containing 2 µM penicillamine or sp-TALP alone, as well as pretreated monolayers, were inseminated with the sperm aliquot pretreated in sp-TALP alone with the same concentration of motile spermatozoa. Unbound spermatozoa were removed 1 h after addition of the sperm, and monolayers were fixed for quantitative analysis.
Experiment 3. (n = 4) addressed the effects of cell-permeant and nonpermeant thiol-reducing agents on sperm release. Monolayers with adhering spermatozoa were incubated for 30 min with 0.1 mM penicillamine, 0.1–1 mM dithiothreitol or reduced glutathione (GSH), or with sp-TALP alone. At the end of treatment, co-cultures were processed as described above for quantitative analysis.
Experiment 4. Experiment 4 (n = 3) was performed to understand if oxidized glutathione (GSSG) was as effective as GSH in releasing adhering spermatozoa. Monolayers with adhering spermatozoa were incubated for 30 min with 0.1 mM penicillamine, 0.1–1 mM GSH or GSSG, or sp-TALP alone. At the end of treatment, co-cultures were processed as described above for quantitative analysis.
Experiment 5. Experiment 5 (n = 3) was designed to study the effect of thiol and non-thiol antioxidants on release of spermatozoa adhering to oviductal monolayers. To this end, co-cultures were treated for 30 min with 0.1 mM penicillamine or with the following antioxidants: 1 mM dimethyl thiourea, 0.5 mM trolox, 2500 U/ml superoxide dismutase, or 120 U/ml catalase. Wells were washed free of unbound spermatozoa and fixed for quantitative analysis.
Experiment 6. Experiment 6 (n = 3) was designed to study the sperm-surface sulfhydryl distribution patterns in the initial sperm suspension (time 0), in unbound and adhering spermatozoa after 1 h of co-incubation, and in spermatozoa released by 30-min treatment with 0.1 mM penicillamine. For this purpose, 100-µl aliquots of sperm suspensions or subpopulations in sp-TALP were incubated with 4 µM IAF for 15 min at room temperature in the dark, then diluted to 10 ml with sp-TALP, centrifuged for 10 min at 180 x g, resuspended in fresh medium, and observed in vivo with a fluorescence microscope. Monolayers grown on gelatin-coated coverslips with adhering spermatozoa were labeled with IAF as stated, extensively washed with sp-TALP, mounted with fresh medium on a glass slide with cells facing up, and observed in vivo with a fluorescence microscope. Ten to twenty fields (depending on sperm concentration) were alternately acquired with phase contrast and fluorescence imaging. Labeling patterns were expressed as percentages through analysis of at least 500 spermatozoa for each condition.
Experiment 7. Experiment 7 (n = 3) was designed to study the acrosomal status of spermatozoa released by thiol-reducing agents. Co-cultures were treated with 0.1 mM penicillamine or 1 mM DTT or GSH for 30 min. At the end of treatment, aliquots of co-culture supernatants were placed on glass slides, air dried, permeabilized in 95% ethanol for 1 h, washed in PBS, incubated for 30 min in the dark with 10 µg/ml fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC; Sigma, Milan) in PBS, washed extensively in PBS, then were mounted and observed with a fluorescence microscope. Ten fields were acquired with filters for fluorescein, and percentages of acrosome-intact spermatozoa were determined by counting at least 1000 spermatozoa per sample.
Experiment 8. Experiment 8 (n = 3) was designed to study the capacitation status of the initial sperm suspension (time 0), of unbound and adhering spermatozoa at 1 h of coincubation, and of spermatozoa released by 30-min treatment with 0.1 mM penicillamine. To this end, sperm suspensions or co-cultures, treated with 100 µg/ml LPC (stock solution, 30 mg/ml in ethanol) or with 0.33% ethanol (control) in sp-TALP for 15 min, were placed on glass slides and/or air dried. Samples were then processed for assessment of acrosomal status with PSA-FITC, as described above. Acrosomal status was evaluated by scoring at least 400 spermatozoa treated with LPC or not for each condition. Capacitation of a given sperm population was represented by the difference between percentages of acrosome-reacted spermatozoa in LPC-treated and in basal populations.
The data are presented as mean ± SD. Overall analysis was performed by the estimate model of ANOVA [30] followed by the Tukey's honestly significant difference test for pairwise comparisons when overall significance was detected. Raw data from experiment 1 and experiments 3–8 were modified by arc sine transformation to normalize data.
Thiol-Reducing Agents Induce the Release of Spermatozoa Adhering to Fallopian Tube Epithelium
Experiment 1 (n = 5) was designed to study the effect of the thiol-reducing agents D-penicillamine, DL-penicillamine, beta mercaptoethanol, and cysteine on release of spermatozoa adhering to oviductal monolayers. All agents, at both 0.1 and 1 mM concentrations, were able to induce a significant release of adhering spermatozoa compared to control (Fig. 1; P < 0.001). All thiols tested were prepared at the time of treatment, since preliminary experiments showed that their sperm-releasing activity was totally lost within a few hours after solubilization. As observed previously during sulfated glycoconjugate-induced release (unpublished observations), spermatozoa adhering to plastic or gelatine-coated coverslips were not released by any of the thiols tested. With regard to the effectiveness of thiols tested, D- and DL-penicillamine were equally effective and represented the most powerful sperm-releasing agents among those tested (P < 0.001). In fact, their activity was maximal at 0.1 mM concentration (90% sperm release), and only a slight, nonsignificant increment was observed at 1 mM concentration. Cysteine at 1mM concentration was more effective than beta mercaptoethanol (P < 0.001). Released spermatozoa displayed rapid linear motility and, different from heparin-released sperm, showed no sign of agglutination (not shown). The sperm-releasing action of thiol-reducing agents, as previously shown for sulfated glycoconjugates, was not related to the oviductal culture stage. In fact, penicillamine at 0.1 mM concentration was equally effective on spermatozoa adhering to monolayers and on explants cultured as previously reported [29].
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Thiol-Reducing Agents Act on Spermatozoa
Results of Experiment 1 demonstrated that thiol-reducing agents were powerful inducers of sperm release. However, this sperm-releasing effect might have been exerted directly on spermatozoa and/or on oviductal cells. In Experiment 2, spermatozoa or oviductal cells were pretreated for 30 min with 100 µM penicillamine and, after washing, were co-incubated with counterpart cells. In this experiment, two controls were used: the first for oviductal cell pretreatment in sp-TALP alone; the second for sperm pretreatment with 2 µM penicillamine (i.e., with the residual concentration present in the well inseminated with pretreated spermatozoa). The quantitative analysis of sperm adhesion (Fig. 2) clearly demonstrated that monolayer pretreatment did not affect sperm adhesion, whereas sperm pretreatment induced a reduction of sperm adhesion comparable to that observed in experiment 1 (P < 0.001).
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Ability of the Nonpermeant Thiol-Reducing Agent GSH to Induce Sperm Release
Thiol-reducing agents used in previous experiments were all cell permeant. The aim of experiment 3 was to determine whether the releasing action of thiols on spermatozoa was exerted on intracellular or on surface molecules. To this end, adhering spermatozoa were treated with cell-permeant and cell-nonpermeant thiol-reducing agents. Data in Figure 3 show that the permeant thiol DTT had a releasing effect quantitatively similar to penicillamine and, more interestingly, that the nonpermeant thiol GSH also induced a significant sperm release (P < 0.001).
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GSSG Does Not Induce Sperm Release
Previous experiments showed that both permeant and nonpermeant thiols were able to induce the release of spermatozoa adhering to the tubal epithelium in vitro. To determine whether the action of thiols was due to their reduced condition, oxidized (GSSG) and reduced glutathione (GSH) were tested for their ability to release adhering spermatozoa in experiment 4. For this purpose, monolayers with adhering spermatozoa were incubated with 0.1 mM penicillamine, 0.1–1 mM GSH or GSSG, or sp-TALP alone. Data in Figure 4 show that GSSG even at 1 mM concentration was unable to induce release of spermatozoa, thus indicating that the reduced state is necessary for thiols to have sperm-releasing action (P < 0.001).
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Non-Thiol Antioxidants Do Not Induce Sperm Release
The aim of Experiment 5 (n = 3) was to understand whether non-thiol antioxidants were able to elicit release of spermatozoa adhering to the fallopian tube epithelium in vitro. To this end, co-cultures were treated with the following enzymatic and nonenzymatic antioxidants: 1 mM dimethyl thiourea, 0.5 mM trolox, 2500 U/ml superoxide dismutase, and 120 U/ml catalase. Parallel wells were treated with 0.1 mM penicillamine as a control for sperm release. Data in Figure 5 demonstrate that only catalase had a slight but nonsignificant sperm-releasing effect.
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IAF-Labeled Spermatozoa Do Not Adhere to Oviduct
Experiment 6 (n = 3) was designed to study distribution patterns of sulfhydryl on the sperm surface in the initial sperm suspension (time 0), on unbound and adhering spermatozoa at 1 h of co-incubation, and in spermatozoa released by 30-min treatment with 0.1 mM penicillamine. For this purpose, spermatozoa were labeled in vivo with the nonpermeant, sulfhydryl-labeling reagent IAF, washed with fresh sp-TALP, and then observed with a fluorescence microscope. Spermatozoa processed for localization of surface sulfhydryls exhibited four labeling patterns (Fig. 6): 1) totally unlabeled (UNL); 2) homogeneous labeling of head, middle piece, and part of the tail (H); 3) labeling of post-acrosomal region and tail (PA); 4) labeling of a final portion of or the whole tail (T). Data in Figure 7 demonstrate the following: 1) about 55% of spermatozoa in the initial suspension were unlabeled; 2) most (about 85%) adhering spermatozoa at 1 h of co-culture were unlabeled (Fig. 8), showing that spermatozoa devoid of surface sulfhydryls adhered to the oviduct (adhering 1 h vs. time 0, P < 0.05); 3) the great majority (about 82%) of spermatozoa unable to adhere were labeled (unbound 1 h vs. adhering 1 h, P < 0.001; unbound vs. time 0, P < 0.001); and 4) the proportion of labeled spermatozoa increased after release by means of thiol-reducing agents, although not significantly (adhering 16% ± 3% vs. released 28% ± 4%). Analysis of labeling patterns in the four populations tested (Fig. 9) demonstrate that labeled adhering spermatozoa exposed surface sulfhydryls only at the extra-acrosomal regions (see also Fig. 8), whereas after release, a proportion of spermatozoa displayed a homogeneous pattern of labeling.
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Spermatozoa Released by Thiol-Reducing Are Acrosome Intact
Experiment 7 (n = 3) was performed to assess the acrosomal status of spermatozoa released by thiol-reducing agents. Co-cultures were treated for 30 min with 0.1 mM penicillamine, 1 mM DTT, or 1 mM GSH. At the end of treatment, released spermatozoa were treated with PSA-FITC assess acrosomal status. Results shown in Figure 10 demonstrate that the great majority of released spermatozoa were acrosome intact, indicating that the releasing action of thiol-reducing agents was not due to an eventual triggering of the acrosome reaction.
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Spermatozoa Released by Thiol-Reducing Agents Are Capacitated
Previous papers on release of spermatozoa adhering to the fallopian tube epithelium in vitro have indicated that this event is due to changes in sperm affinity toward the apical membrane of oviductal cells and that such remodeling represents one of the earliest events of capacitation [4, 8, 29]. The aim of experiment 8 (n = 3) was to determine whether sperm release induced by thiol-reducing agents was associated with capacitation. For this purpose, the acrosomal status of spermatozoa was assessed with PSA-FITC in the initial sperm suspension (time 0), in unbound and adhering spermatozoa at 1 h of coincubation, and in spermatozoa released after 30-min treatment with 0.1 mM penicillamine, and treated or not with 100 µg/ml LPC in sp-TALP. Data (Fig. 11) indicate that uncapacitated spermatozoa adhere to the fallopian tube epithelium in the initial suspension and this subpopulation is maintained in the uncapacitated state (adhering vs. unbound, P < 0.001). Moreover, release induced by the thiol-reducing agent penicillamine was associated with a rapid change toward a capacitated state (adhering vs. released, P < 0.001).
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The main results demonstrated that permeant as well as nonpermeant thiol-reducing agents are able to trigger the release of spermatozoa adhering to the fallopian tube epithelium in vitro. To our knowledge, this is the first evidence that this particular class of antioxidants, whose presence in the oviductal fluid has been reported in different species [26, 31–33], may represent a physiological signal that induces the detachment of selected and stored spermatozoa, at least in the bovine species. Pretreatment of spermatozoa with thiol-reducing agents markedly reduced cell-cell adhesion, whereas treatment of oviductal monolayers with the same agents did not; thus, the action of the thiol-reducing agents is exerted directly on spermatozoa. Moreover, GSH, a nonpermeant thiol [34] physiologically present in bovine oviductal fluid, whose concentration peaks at ovulation [26], was also able to induce sperm release, indicating that thiol-induced sperm release may proceed through reduction of sperm surface disulfides to sulfhydryls. The lack of effect of dimethylthiourea, trolox, superoxide dismutase, and catalase on sperm release indicates that this process requires reduction of sperm surface disulfides, whereas other enzymatic and nonenzymatic non-thiol antioxidants physiologically present in the oviductal fluid [15, 26] may contribute to maintaining a correct balance between oxidants and antioxidants to protect gametes and embryos. It is worth mentioning that bovine oviductal fluid contains a specific form of catalase that reaches its maximal activity just before ovulation and is able to associate with the acrosomal region of the sperm surface [26, 35]. The possible involvement of oviduct-specific catalase in sperm release from the oviductal reservoir may warrant further investigation.
Although capacitation is considered an oxidative process, in the human it has been associated with a marked, time-dependent increase of sperm membrane sulfhydryl groups exposed to the extracellular space [36]. Interestingly, both sulfhydryl-targeted reagents and disulfide reductants are able to induce human sperm capacitation [37]. In bovine spermatozoa, cryopreservation has been shown to induce a premature capacitation [38] that is also accompanied by a marked increase in surface protein sulfhydryls, which can be prevented by both GSH and GSSG [39]. However, under our conditions, treatment of co-cultures with GSSG did not induce release of adhering spermatozoa, confirming again the hypothesis that GSH acts through reduction of sperm surface disulfides to sulfhydryls.
To determine whether the sperm-releasing action of thiols was due to the reduction of sperm-surface disulfides to sulfhydryls, the initial sperm suspension, the adhering and the unbound sperm subpopulation, as well as the spermatozoa released by penicillamine were challenged with the nonpermeant sulfhydryl-labeling reagent iodoacetamide-fluorescein. Results demonstrated that the initial sperm suspension was composed of spermatozoa devoid of surface sulfhydryls and spermatozoa sulfhydryls exposed at the level of different surface domains [39]. Interestingly, mainly unlabeled spermatozoa from the initial suspension adhered to the fallopian tube epithelium, and adhering spermatozoa never had detectable sulfhydryls on the sperm rostral region (i.e., the region involved in binding to the tubal epithelium [4]). The sperm subpopulation unable to adhere was composed principally of spermatozoa that expose sulfhydryls, whereas the percentage of labeled spermatozoa after thiol-induced release increased, although not significantly compared to adhering spermatozoa. Overall, results demonstrate that 1) spermatozoa that adhere do not reveal sulfhydryls on their surface, 2) adhering spermatozoa are maintained in this condition, whereas 3) thiol-induced release proceeds through reduction of sperm surface disulfides. The lack of increase of IAF labeling in the majority of the released sperm subpopulation may indicate that only one or few sperm surface proteins are responsible for redox modulation of the transient sperm-oviduct adhesion, and their reduction could be detected only through use of biochemical tools. Recently, subtle and reversible redox modifications have emerged as physiologic mechanisms for post-transitional modulation of protein functions. In fact, sulfhydryl-disulfide oxidoreduction has been shown to play a key role in the redox regulation of cell signaling and gene expression [40–43]. Although attention has been focused on intracellular targets of redox regulation, several papers have addressed the role of surface thiols in the modulation of adhesion proteins [44–46]. Therefore, redox modulation of sperm release from the fallopian tube epithelium may be due to reduction of specific sperm-surface proteins directly or indirectly involved in adhesion.
Several reports have indicated that capacitation modulates sperm adhesion and release from oviductal cells in a number of species [2, 4, 7, 29]. In cattle, analysis of adhering sperm intracellular calcium and protein tyrosine phosphorylation during heparin-induced sperm release led us to suggest that detachment of spermatozoa from the tubal epithelium may represent one of the earlier events recognizable during capacitation [8].
Analysis of capacitation status through lysophosphatidyl choline (LPC)-induced acrosome reaction showed that oviductal adhesion selects uncapacitated spermatozoa and maintains them in this condition. As expected, spermatozoa unable to bind to the tubal epithelium were capacitated. Finally, thiol-induced release was accompanied by a marked increase in the percentage of capacitated spermatozoa compared to the adhering sperm subpopulation. Therefore, as previously demonstrated for sulphated glycoconjugate-induced release, thiol-induced release of adhering spermatozoa is associated with the process of capacitation. The action of thiols closely resembles the effect of heparin and other sulphated glycoconjugates previously demonstrated to powerfully induce sperm release [29], showing that different and apparently unrelated molecules naturally occurring in the bovine oviductal fluid act as releasing signals for spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.
Recent papers indicate that three heparin-binding proteins of bovine seminal plasma (BSPs), PDC-109, BSP-A3, and BSP-30kDa, that coat epididymal spermatozoa at ejaculation are responsible for the transient sperm adhesion to the fallopian tube epithelium [47, 48]. Heparin binding to BSPs triggers capacitation and the loss of affinity for the epithelium. Interestingly, the presence of a heparin binding site and of four disulfide bridges in each BSP [49] raises the attractive hypothesis that heparin and thiol-reducing agents represent signals acting, through different mechanisms, on sperm surface BSPs.
The recognition of thiols as powerful inducers of sperm release and the demonstration that their action occurs through a reduction of sperm surface disulfides to sulphydryls contribute to the understanding of sperm selection and storage within the female tract and provide new opportunities in the search for still poorly known sperm surface molecules involved in adhesion and release from the fallopian tube epithelium.
FOOTNOTES
1Supported by P.R.I.N. grant 2004. 
Correspondence: 2R. Talevi, Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli "Federico II," Complesso Universitario di Monte S Angelo, Via Cinthia, 80126 Napoli, Italy. FAX: 81 679233; e-mail: riccardo.talevi{at}unina.it
Received: 24 July 2006.
First decision: 30 October 2006.
Accepted: 19 December 2006.
REFERENCES
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